The present invention relates to spectroscopic imaging of heterogeneous light scattering tissue, and more particularly, but not exclusively, relates to in vivo imaging by mapping a fluorescence characteristic of the tissue.
The early detection of disease promises a greater efficacy for therapeutic intervention. In recent years, noninvasive techniques have been developed which have improved the ability to provide a reliable and early diagnosis of various afflictions by detecting biochemical changes in the tissue of a patient. For example, Magnetic Resonance Imaging (MRI) has successfully monitored the relaxation of spin states of paramagnetic nuclei in order to provide biomedical imaging and biochemical spectroscopy of tissues. Unfortunately, the complexity and expense of MRI diagnostics limit its availability—especially as a means of routine monitoring for disease.
Another powerful analytical technique with an increasing number of applications in the biological sciences is fluorescence spectroscopy. Applications of fluorescence spectroscopy include biomedical diagnostics, genetic sequencing, and flow cytometry. As exemplified by U.S. Pat. Nos. 5,421,337 to Richards-Kortum et al. and U.S. Pat. No. 5,452,723 to Wu et al., several investigators have suggested various procedures to differentiate diseased and normal tissues based on fluorescence emissions through noninvasive external measurements or minimally invasive endoscopic measuring techniques. Unfortunately, these procedures generally fail to provide a viable spatial imaging procedure. One reason imaging based on fluorescence has remained elusive is that meaningful relational measurements of fluorescence characteristics from a random, multiply scattering media, such as tissue, are difficult to obtain. For example, fluorescent intensity, which is a function of the fluorescent compound (or fluorophore) concentration or “uptake” is one possible candidate for imaging; however, when this property is used in an optically dense medium, such as a particulate (cell) suspension, powder, or tissue, the local scattering and absorption properties confound measured fluorescent intensities.
Besides intensity, other properties of selected fluorophores such as fluorescent quantum efficiency and lifetime are also sensitive to the local biochemical environment. As used herein, “fluorescent quantum efficiency” means the fractional number of fluorescent photons emitted for each excitation photon absorbed or the fraction of decay events which result in emission of a fluorescent photon. “Fluorescent lifetime,” as used herein, is defined as the mean survival time of the activated fluorophore or the mean time between the absorption of an excitation photon and emission of a fluorescent photon. Like intensity, measurement of these fluorescence characteristics is often limited to well-defined in vitro applications in the research laboratory or in flow cytometry where issues such as scattering, absorption, and changing fluorophore concentrations can be controlled or measured. Moreover, these limitations generally preclude meaningful fluorescence-based imaging of hidden tissue heterogeneities, such as tumors or other diseased tissue regions, which cannot be detected by visual inspection.
With the development of techniques to interrogate tissues using fluorescence in the near-infrared red (NIR) wavelength regime, noninvasive detection of diseased tissues located deep within normal tissues may also be possible since NIR excitation and emission light can travel significant distances to and from the tissue-air interface. U.S. Pat. Nos. 5,213,105 to Gratton et al. and U.S. Pat. No. 5,353,799 to Chance are cited as further background concerning NIR interrogation. As in the case of MRI, x-ray, CT, and ultrasound imaging modalities, there is a potential to enhance NIR fluorescence imaging techniques with contrast agents. Typically, contrast agents for in vivo imaging have depended upon preferential uptake into diseased tissue to provide the desired imaging enhancement by absorbing the interrogating radiation. The light absorbing tissue provides an enhanced spatial variation in measured intensity of the radiation to improve image contrast. In the case of a fluorescent contrast agent, the intensity of fluorescent light emitted in response to the absorption may provide this intensity variation. Generally, the larger the difference in spatial variation, as artificially imposed by a contrast agent, the more improved the reconstructed image of interior tissues. Nonetheless, the effectiveness of exogenous contrast agents depends greatly upon the selectivity of the agent for the tissue region of interest. Unfortunately, targeted and site specific delivery of drugs and contrast agents has historically been a limiting factor in both therapeutics and diagnostic imaging. Consequently, additional mechanisms for inducing contrast that are not dependent solely upon tissue selectively of the agent would be advantageous.
Thus, a need remains for a technique to noninvasively image multiply scattering tissue based on one or more fluorescence characteristics. Preferably, this technique includes the implementation of exogenous contrast agents with image-enhancing properties beyond preferential absorption of the interrogating radiation. The present invention satisfies this need and provides other advantages.